New cyclopeptides

ABSTRACT

Cyclopeptides having antitubercular activity derived from antibiotics of the formula: R-cyclopeptide A wherein cyclopeptide A designates a nonapeptide residue of the formula: IN WHICH MePro signifies L-trans-4-methyl-proline; MeThr signified L-N-methylthreonine; MeVal signifies L-N-methylvaline; MeLeu signifies D-N-methylleucine; Pro signifies L-proline; Gly signifies glycine, and Leu signifies L-leucine, and R represents hydrogen or an alkanoyl, alkenoyl, alkadienoyl, alkoxycarbonyl, aroyl, aralkanoyl, arylsulphenyl, arylsulphinyl, aryl sulphonyl, cycloalkylcarbonyl, heterocyclyclcarbonyl or heterocyclylakanoyl radical, or a residue of a linear or cyclic peptide radical attached through a carbonyl group to the nitrogen atom of the Ltrans-4-methyl-proline grouping the the side chain of the cyclopeptide A, and non-toxic acid addition and quaternary ammonium salts thereof, possess antitubercular activity and activity against gram-positive and gram-negative microorganisms.

United States Patent [191 Jolles [5 1 March 6, 1973 NEW CYCLOPEPTIDES[75] Inventor: Georges Jolles, Sceaux (Hauts-de- Related US. ApplicationData [63] Continuation-impart of Ser. No. 770,436, Oct. 24,

1968, abandoned.

[30] Foreign Application Priority Data Primary ExaminerElbert L. RobertsAttorney-Stevens, Davis, Miller & Mosher [5 7 1 ABSTRACT Cyclopeptideshaving antitubercular activity derived from antibiotics of the formula:R-cyclopeptide A wherein cyclopeptide A designates a nonapeptide residueof the formula:

in which MePro signifies L-trans-4-methyl-proline; MeThr signifiedL-N-methylthreonine; MeVal signifies L-N-methylvaline; MeLeu signifiesD-N-methylleucine; Pro signifies L-proline; Gly signifies glycine, andLeu signifies L-leucine, and R represents hydrogen or an alkanoyl,alkenoyl, alkadienoyl, alkoxycarbonyl, aroyl, aralkanoyl, arylsulphenyl,arylsulphinyl, aryl sulphonyl, cycloalkylcarbonyl, heterocyclyclcarbonylor heterocyclylakanoyl radical, or a residue of a linear or cyclicpeptide radical attached through a carbonyl group to the nitrogen atomof the L-trans-4-methylproline grouping the the side chain of thecyclopeptide A, and non-toxic acid addition and quaternary ammoniumsalts thereof, possess antitubercular activity and activity againstgram-positive and gram-negative microorganisms.

4 Claims, No Drawings NEW CYCLOPEPTIDES This invention relates to newtherapeutically useful cyclopeptides and acid addition and quaternaryammonium slats thereof, to processes for their preparation andpharmaceutical compositions containing them. This application is aContinuation-impart of my application Ser. No. 770,436 filed October 24,1968 and entitled New Cyclopeptides, and now abandoned.

The new cyclopeptides of the present invention are those of the generalformula:

R-cyclopeptide A 1 wherein cyclopeptide A designates a nonapeptideresidue of which the structure corresponds to the formula:

bonyl, heterocyclylcarbonyl or heterocyclylalkanoyl radical, or aresidue of a linear or cyclic peptide radical attached through acarbonyl group to the nitrogen atom of the L-trans-4-methyl-prolinegrouping of the side chain of the cyclopeptide A, and acid addition andquaternary ammonium salts thereof.

In the foregoing definition of R, the alkyl, alkenyl or alkadienylgroups of the alkanoyl, alkenoyl, alkadienoyl, aralkanoyl orheterocyclylalkanoyl radicals are linear or branched chains containingone to 50 carbon atoms (preferably one to 18 carbon atoms), optionallysubstituted by one or more halogen atoms or amino or alkylthio groupscontaining one to five carbon atoms, and the alkyl groups ofalkoxycarbonyl radicals containing one to carbon atoms and may besubstituted by a dialkylamino group in which each alkyl portion containsone to five carbon atoms, the aromatic groups of the aroyl, aralkanoyl,arylsulphenyl, arylsulphinyl or arylsulphonyl radicals represent abenzene or naphthalene nucleus which is optionally substituted by one ormore alkyl, hydroxy, benzoyl, dialkylamino, alkanoylamino or nitrogroups, with the alkyl and the alkyl portions of the other radicalscontaining one to 12 carbon atoms, the heterocyclic groups of theheterocyclylcarbonyl or heterocyclylalkanoyl represent mononuclear orpolynuclear heterocycles containing one or more hetero atoms selectedfrom radicals cycloalkyl radicals having five or six carbon atomsoptionally substituted by one or more amino groups, the

residues of the linear or cyclic peptide radicals contain 2 to 15aminoacids, and the amino groups present as 5 substituents in the alkyl,alkenyl or cycloalkyl portions Y of the radicals defined above, and theamino groups of the linear or cyclic peptide radicals, may be optionallysubstituted by one or more alkyl, alkanoyl, aralkyl, alkoxycarbonyl,aralkoxycarbonyl or alkoxyaralkoxycarbony] groups, with the alkylradicals and the alkyl portions of the other radicals containing one to30 carbon atoms.

Examples of radicals represented by the symbol R areN-a-acetyLN-e-benzyloxycarbonyl-lysyl, N-benzyl- N-methyl-valyl,Nbenzyloxycarbonylvalyl, N- benzyloxycarbonyl-glycyl, N-benzylsarcosyl,N- decanoyl-valyl, N-benzyl-N-methyl-methionyl, N

benzyloxycarbonyl-phenylglycyl, N-hexyLN-methylvalyl,N-benzyl-N-methyl-alanyl, N-benzyl-N-methylphenylalanyl, N-hexylvalyl,N-benzyl-N-methyl-leucyl, N,N-diheptylglycyl, N-t.butyloxycarbonybvalyl,N-apalmitoyl-N-e-benzyloxycarbonyl-lysyl, N-a'pelargo- 2 5nyl-Ne-benzyloxycarbonyl-lysyl,N-a-6-methyloctanoyl-Ny-benzyloxycarbonyl-aq diaminobutyrylthreonyl-N-y-benzyloxycarbonyl-ag-diaminobutyryl-N-methyl-valyl, N-stearoyl-valyl, N-stearoyl-N-methylvalyl, methionyl,N-acetyl-lysyl, valyl, N-a-6-methyloctanoyl-a,-diaminobutyryl-threonyl-a,-y-diaminobutyryl-N-methyl-valyl,N-palmitoyHysyl, N-pelargonyllysyl,N-methyl-p-dimethylaminophenylalanyl, undecl0-enoyl,1-methyl-4-nitro-2-pyrrolylcarbonyl, palmitoyl, undecanoyl, dodecanoyl,6-methyloctanoyl, benzoyl, isonicotinoyl, salicyloyl, decanoyl,4-pmethoxybenzyloxycarbonylamino-cyclohexylcarbonyl,(l0-methyl-3phenthiazinyl)acetyl, 2-(3-benzoylphenyl)propionyl,N-a-6methyloctanoyl-Ny-benzyloxycarbonyl-og'y-diaminobutyryl-threonyl-N-y-benzyloxycarbonyl-a,y-diaminobutyryl,N-a-paImitoyl-N-ybenzyloxycarbonyl-a,-y-diaminobutyryl, N-a-6-rnethyloctanoyl-N-y-benzyl-oxycarbonyl-a,-ydiaminobutyryl, heptanoyl,stearoyl, n-butyryl, sorboyl, l l-l pyrrolidinyl-undecanoyl, 2-1pyrrolidinyl-hexanoyl, 6-1 pyrrol-idinyl-hexanoyl, ll-diethylaminoundecyloxycarbonyl, decyloxycarbonyl, N-a-6-methyloctanoyl'aq-diaminobutyryl-threonyl-a,-ydiaminobutyryl,N-a-6-methyloctanoyl-a,'y-diaminobutyryl, 4-amino-cyclohexylcarbonyl,trichloroacetyl, onitrophenylsulphenyl, l-dimethylamino-S-naphthalenesulphonyl, toluene-p-sulphonyl, pacetylaminobenzenesulphonyl,N-undecyl-N-methylvalyl, N-dodecyl-Nmethyl-valyl,N-tridecyl-N-methylvalyl and Ntetradecyl-N-methyl-valyl.

1. According to a feature of the invention, the cyclopeptide of generalFormula (I) wherein R represents a hydrogen atom, i.e., cyclopeptide A,

is obtained by hydrolysis of the antibiotic designated by the number11,072 R.P. by treatment with an acid in an organic or aqueous-organicsolution.

nitrogen, oxygen and sulphur atoms (e.g., pyrrolyl, pyrrolidinyl andphenthiazinyl) and optionally substituted by one or more alkyl or nitrogroups, the cycloalkyl groups of the cycloalkylcarbonyl radicalsrepresent Antibiotic 11,072 R.P. and its preparation have been describedin British Pat. No. 966,124 granted on an application filed byRhone-Poulenc SA. on June 4, i962.

It has been established that this antibiotic has the structure shownbelow:

CH3 CH3 CH(CHa)2 (III) Inorganic acids (for example hydrochloric andsulphuric acids) or organic acids (for example trifluoroacetic acid) atvarious concentrations are suitable for hydrolysis of 1 1,072 R.P., buthydrochloric acid is preferably used at normalities of between 1N and6N. Solvents which may generally be employed in the process are alcoholscontaining one to four carbon atoms, ethers such as dioxan ortetrahydrofuran, esters such as ethyl acetate, or a mixture of two ormore of such solvents. Methanol containing anhydrous hydrogen chlorideis preferably used for the hydrolysis.

The concentration in the medium of antibiotic 1 1,072 R.P. may vary from1 to 20 percent.

Hydrolysis is carried out at a temperature of between and 50 C. and thereaction time, which is a function of the temperature, may vary fromhours to 1 hour. The process is generally carried out at C. for 2 hours.

Cyclopeptide A thus prepared may optionally be purified by the usualphysical or chemical methods, for

example by chromatography.

2. According to another feature of the invention, the cyclopeptides ofgeneral Formula (I) wherein R represents a radical R as hereinbeforedefined are obtained from cyclopeptide A by condensation with an acidR'-OH, or a reactive derivative thereof, by any of the known methodsemployed in peptide chemistry.

Examples of suitable condensation methods are those using a carbodiimidesuch as dicyclohexylcarbodiimide, the azide method, the activated estermethod, the mixed anhydride method and the acid chloride method. Inthese processes, free functional groups in the radical R which must notparticipate in the reaction are protected by groups capable of beingsubsequently removed by procedures which do not affect the rest of themolecule. Residues which can be removed by hydrolysis or by reductionare generally employed for the purpose. The amine group is preferablyprotected by alkoxycarbonyl groups, e.g., t.- butoxycarbonyl,aralkoxycarbony] groups, e.g., benzyloxycarbonyl, or aralkyl groups,e.g., benzyl.

More particularly, when an acid of the general formula:

R"OH (IV) (wherein R" has the same significance as R defined above withthe exclusion of alkoxycarbonyl, arylsulphenyl, arylsulphinyl andarylsulphonyl radicals) is used, the reaction is effected in an organicsolvent, such as ethyl acetate, dimethylformamide, acetonitrile ormethylene chloride, at a temperature of between 0 More particularly,when an azide of the general formula:

3 (wherein R is as hereinbefore defined) is used, the reaction iseffected in an organic solvent such as ethyl acetate, optionally in thepresence of an organic base such as triethylamine and at a temperatureof between 1 5 C. and +25 C.

The azides of general Formula (V) are generally prepared by reaction ofan alkali metal nitrite with the corresponding hydrazide in an acidmedium.

More particularly, when a reactive ester of the general formula:

R"X (VI) (wherein R" is as hereinbefore defined, and X represents aphenol residue or a residue of a nitrogencontaining N-hydroxylatedheterocyclic compound which activates the carbonyl group in the radicalR to which it is attached, for example N-hydroxysuccinimide,p-nitrophenol, 2,4,5-trichlorophenol or I- hydroxypiperidine) is used,the reaction is effected in an organic solvent,'e.g., ethyl acetate ordimethylformamide, in the presence of a carbodiimide, e.g.,dicyclohexylcarbodiimide, at a temperature of between l5 C. and +25 C.in the optional presence of an organic base, e.g., triethylamine.

the reactive esters of Formula (VI) are generally prepared in situaccording to methods known per se.

More particularly, when an acid anhydride of the general formula:

R"--OCO--O--R (Vll) (wherein R is as hereinbefore defined, and Rrepresents an alkyl radical containing one to five carbon atoms) isused, the reaction is effected in an organic solvent such as methylenechloride at a temperature between l5 C. and +20 C., optionally in thepresence of an organic base such as triethylamine.

The mixed anhydrides of general Formula (Vll) are generally prepared insitu by reaction of an alkyl chloroformate, e.g., ethyl or isobutylchloroformate, with an acid of general Formula (IV) in an organicsolvent, e.g., methylene chloride, in the presence of an organic base,e.g., triethylamine, and at a temperature of about-10C.

More particularly, when an acid chloride of the general formula:

R-Cl (Vlll) (wherein R" is as hereinbefore defined) is used, thereaction is effected in an organic solvent, e.g.,

methylene chloride, optionally in the presence of an organic base, e.g.,triethylamine, and at a temperature of between 1 5 C. and +25 C.

More particularly, for the preparation of cyclopeptides of generalFormula (I) wherein R represents an optionally substitutedalkoxycarbonyl radical as hereinbefore defined, cyclopeptide A isreacted with a chloroformate of the general formula:

wherein R' represents an alkyl radical containing one to carbon atomswhich is optionally substituted by one dialkylamino radical with eachalkyl group thereof containing one to five carbon atoms, in an organicsolvent, e.g., methylene chloride, in the presence of an organic base,e.g., triethylamine, and at a temperature of between l5 C. and +30 C.

More particularly, for the preparation of cyclopeptides of generalFormula (I) wherein R represents an arylsulphenyl, arylsulphinyl orarylsulphonyl radical as hereinbefore defined, cyclopeptide A is reactedwith a compound of the general formula:

R"S(O),,-Cl ()0 wherein R represents a benzene or naphthalene nucleuswhich is optionally substituted by one or more alkyl, hydroxy, benzoyl,dialkylamino, alkanoylamino or nitro groups, and n represents 0, l or 2,in an organic solvent, e.g., ethyl acetate or methylene chloride, in thepresence of an organic base, e.g., triethylamine, and at the temperatureof between l5 C. and C.

3. A modification of the process according to (2) above comprisesreacting an acid or an acid derivative of Formula (IV), (V), (VI),(VII), or

(VIII) with a cyclopeptide of the general formula:

saii as 'snsgtnn NHRa (x1) wherein R and R are the same or different andeach represents a hydrogen atom or an alkyl radical containing one tofive carbon atoms, under the conditions described in respect of process(2).

Where, in the processes described under cfiand 3 the radicals R", R andR of the compounds of Formulas (IV), (V), (VI), (VII), (VIII), (IX) and(X) contain protective groups, these protective groups are thereafterremoved by methods known per se, such as catalytic hydrogenation or acidhydrolysis.

The new cyclopeptides of general Formula.(l) may optionally be purifiedby physical methods (such as chromatography) or chemical methods (suchas the formation of salts which are soluble in water, filtration andlyophilization of the resulting solution, followed by decomposition ofthe resulting product).

The cyclopeptide bases of general Formula (I) may be converted into acidaddition salts or quaternary ammonium salts depending on the nature ofthe substituent R. The acid addition salts may be obtained by reactionof the cyclopeptides with acids in appropriate solvents; generally, thebase is solubilized in water by adding the theoretical amount of acidand the resulting solution is lyophilized. The quaternary ammonium slatsmay be obtained by reaction of the cyclopeptide bases with esters,optionally in an organic solvent, at room temperature or more rapidlywith gentle heating.

The cyclopeptide compounds of Formula (I) and their acid addition andquaternary ammonium salts possess useful therapeutic properties. Theyare antibiotic agents which have a powerful anti-tubercular activityand, furthermore, have a good activity against gram-positive andgram-negative microorganisms.

They have given good results in anti-tubercular activity tests in vitroand in vivo. They have inhibited the multiplication of virulenttubercular bacilli (human strains such as H Rv, bovine strain andvarious mutants of the latter). The activity in vitro is determined bythe method of dilutions in Dubos medium. Under these conditions theminimum inhibitory concentration of the cyclopeptides is between 0.005and l ug./ml. The activity in vivo is determined on experimentallyinfected mice treated with the cyclopeptides for 3 weeks from the dayfollowing the infection. By comparing the survival times of treated anduntreated mice, the minimum effective doses can be determined; they arebetween 50 and 300 mg./kg. p.o.

Of special interest are the cyclopeptides of general Formula (I) inwhich R represents an alkanoyl or alkenoyl radical which is optionallysubstituted by an amino or alkylthio group, or a linear peptide radical,any amino group present being optionally substituted by one or twoalkyl, alkanoyl, aralkyl, alkoxycarbonyl or aralkoxycarbonyl groups. Ofthis class those cyclopeptides, wherein R represents an alkanoyl radicalsubstituted by an amino group or a linear peptide radical, with theamino group(s) present being optionally substituted by one or two alkyl,alkanoyl, aralkyl, alkoxycarbonyl or aralkoxycarbonyl groups, arepreferred. Of outstanding interest are those cyclopeptides of generalFormula (I) wherein R represents the valyl or methionyl group optionallysubstituted on the nitrogen atom by alkyl (preferably methyl and/or analkyl group containing ll to 18 carbon atoms), aralkyl (preferablybenzyl) or alkanoyl (e.g., stearoyl) groups. Of greatest interest are(Nbenzyl-N-methyl-DL-valyl)- cyclopeptide A, (N-benzyl-Nmethyl-L-valyl)-cyclopeptide A, (N-benzyl-N-methyl-D-valyl)- cyclopeptide A,(N-benzyl-Nmethyl-L-methionyl)- cyclopeptide A,(N-stearoyl-L-valyl)-cyclopeptide A and(N-dodecyl-N-methyl-D-valyl)-cyclopeptide A especially the first andlast compounds and acid addition and quaternary ammonium salts thereof.

For therapeutic purposes, the cyclopeptides of general Formula (I) maybe employed as such or in the form of non-toxic acid addition salts,i.e., salts containing anions which are relatively innocuous to theanimal organism in therapeutic doses of the salts (for examplehydrochlorides, sulphates, nitrates, phosphates, acetates, propionates,succinates, benzoates, fumarates, maleates, tartrates,methanesulphonates, benzenesulphonates, theophylline-acetates,salicylates, phenolphthalinates or methylene-bis-fi-hydroxynaphthoates),so that the beneficial physiological properties inherent in thecyclopeptide bases are not vitiated by side-effects ascribable to thoseanions. They may also be employed in the form of non-toxic quaternaryammonium salts obtained by reaction of the cyclopeptides with organichalides, e.g., methyl, ethyl, allyl or benzyl chloride, bromide oriodide, or other reactive esters, e.g., methylor ethylsulphates,benzene-sulphonates or toluene-p-sulphonates.

The following Examples, in which the yields given arerelated to thetheoretical yield, illustrate the invention. The Rf values of theproducts are determined by thin layer chromatography on silica gel.

EXAMPLE 1 The antibiotic peptide 11,072 R.P. (150 g.) obtained asdescribed in British Pat. Specification No. 966,124 is dissolved in 4.5Nhydrochloric acid in methanol (1,320 cc.). The mixture is stirred for 2hours at 20 C. and then concentrated to dryness under reduced pressure(25 mm.Hg). The residue is extracted with methanol (2 X 250 cc.). Themethanol extracts on evaporation yield an amorphous residue which isdissolved in a mixture of chloroform-methanol (95-5 by volume). Theresulting solution is poured into a column 1 l cm in diameter containingsilica gel (2 kg.). Elution is effected with the same solvent. Fractionsof 640 cc. are collected. Fractions 10 to 16 yield thechromatographically pure cyclopeptide A (123.3 g.) in a 95.5 percentyield.

Rf 0.55 (silica gel; 1,2-dichloroethane-methanol,

65-35 by volume) [01],, -62(c= 0.5 methanol). 8

EXAMPLE 2 Cyclopeptide A (3.35 g.) is dissolved in ethyl acetate (70cc.) N-a-Acetyl-N-e-benzyloxycarbonyl L-lysine. (1.13 g.) is added. Themixture is cooled in an ice bath and dicyclohexylcarbodiimide (0.8 g.)is added. The mixture is stirred for 2 hours while externally cooling itin an ice bath, and then overnight at 20 C. Glacial acetic acid drops)is added and the insoluble material formed is then filtered off. Theprecipitate is washed with ethyl acetate, and the organic phases arecombined and then concentrated to dryness under reduced pressure (25mm.l-lg). The residue is taken up in ethyl acetate cc.) and theresulting solution is successively washed with a 2 percent solution ofsodium bicarbonate, then with N hydrochloric acid and finally withdistilled water. The organic phase is dried over anhydrous sodiumsulphate and then filtered. The filtrate is concentrated to drynessunder reduced pressure (25 mm.Hg).

The crude residue (3.14 g.) is charged into a column 3 cm. in diametercontaining silica gel (75 g.). The column is successively eluted withbenzene, 9-1 (by volume) and 5-5 (by volume) mixtures of benzeneethylacetate, ethyl acetate, and 95-5 (by volume), 90-10 (by volume) and75-25 (by volume) mixtures of ethyl acetate-methanol. The fractionseluted with ethyl acetate and with the 95-5 and 90-10 mixtures of ethylacetate-methanol are combined and concentrated to dryness under reducedpressure (25 mm.Hg).

(N-a-Acetyl-N-e-benzyloxycarbonyl-L-lysyl)- cyclopeptide A (2.13 g.) isthus obtained in a 48.5 percent yield.

N'% 12.43 (theory: 12.20)

Rf 0.73 (silica gel; 1,2-dichloroethane-methanol,

8-2 by volume).

The following products are prepared in the same manner, starting withappropriate starting materials: (N-benzyl-N-methyl-L-valyl)-cyclopeptideA C 64.13 (theory 64.30), H 8.53 (theory:

8.67) Rf= 0.67 (silica gel 1,2-dichloroethane-methanol,

8-2 by volume).

(N-benzyl-N-methyl-D-va1yl)-cyclopeptide hydrochloride N 11.50 (theory:11.69) Cl 2.95 (theory:

5 Rf= 0.87 (silica gel 1,2-dichloroethane-methanol,

65-35 by volume).

(N-benzyl-N-methyl-DL-va1yl)-cyclopeptide A methanesulphonate N% 10.59(theory: 11.13) S 2.65 (theory: 2.55)

Rf 0.74 (silica gel l,2-dichloroethane-methanol,

(N-benzylsarcosyl)-cyclopeptide A methanesulphonate N 10.81 (theory:11.52) S 2.89 (theory:

Rf= 0.77 (silica gel 1,2-dichloroethane-methanol,

65-35 by volume). (N-decanoyl-L-valyl)-cyclopeptide A N 11.60 (theory11.56) Rf= 0.83 (silica gel l,2-dichloroethane-methanol,

8-2 by volume). (undec-l0-enoyl)-cyclopeptide A C 64.33 (theory: 64.08)N 10.95 (theory:

1 1.21 Rf 0.58 (silica gel l,2-dichloroethane-methanol,

8-2 by volume). (1-methyl-4-nitro-2-pyrrolylcarbonyl)-cyclopeptide A N13.81 (theory: 13.87) Rf 0.84 (silica gel 1,2-dichloroethane-methanol,

8-2 by volume). (N-benzyl-N-methyl-L-methionyl)-cyclopeptidehydrochloride N 11.62 (theory 11.39) S 3.12 (theory 2.61 Rf 0.65 (silicagel n-butanol-acetic acid-water,

4-1- by volume). (N-benzyloxycarbonyl-L-phenylglycyl)-cyclopeptide A N11.11 (theory 11.42) Rf 0.85 (silica gel 1,2-dichloroethane-methanol,

65-35 by volume). (N-hexyl-N-methyl-L-valyl)-cyc1opeptide hydrochlorideC (for the base) 63.45 (theory 2 63.40) H (for the base) 9.12 (theory9.24) N (for the base) 12.03 (theory: 12.12) Rf 0.53 (silica gell,2-dichloroethane-methanol,

8-2 by volume). 60 (N-benzyl-N-methyl-L-alanyl)-cyclopeptidehydrochloride N 12.20 (theory: 11.98) Cl 3.3 (theory:

3 .03) Rf= 0.85 (silica gel 65-35 by volume).(N-benzyl-N-methyl-L-phenylalanyl)-cyclopeptide A N 1 1.50 (theory:11.58) Rf 0.77 (silica gel l,2-dichloroethane-methanol,

8-2 by volume).

l,2-dichloroethane-methanol,

palmitoyl-cyclopeptide A C 64.45 (theory 65.24) H 9.41 (theory 9.51) N10.30 (theory: 10.53) Rf 0.77 (silica gel 1,2-dichloroethane-methanol,

8-2 by volume). undecanoyl-cyclopeptide A c 64.78 (theory 64.0) H 9.28(theory 9.21) N 10.9 (theory: 11.18) Rf 0.52 (silica gel1,2-dichloroethane-methanol,

82 by volume). dodecanoyl-cyclopeptide A N %=11.1 (theory: 11.05) Rf0.78 (silica gel 1,2-dichloroethane-methanol,

82 by volume). [L(+)-6-methyloctanoylLcyclopeptide A C 63.63 (theory63.41) H 8.50 (theory:

9.03) I N 11.12 (theory 11.47) Rf 0.77 (silica gel1,2-dichloroethane-methanol,

82 by volume). benzoyl-cyclopeptide A N 11.88 (theory: 11.86) Rf 0.85(silica gel 1,2-dichloroethane-methanol,

65-35 by volume). isonicotinoyl-cyclopeptide A N 13.14 (theory: 13.17)R1 0.83 (silica gel 1,2-dichloroethane-methanol,

65-35 by volume). salicyloyl-cyclopeptide A N%=11.97 (theory: 11.68) Rf0.80 (silica gel 1,2-dichloroethane-methanol,

65-35 by volume). decanoyl-cyclopeptide A C 63.98 (theory 63.69) H 9.41(theory:

9.15) Rf 0.70 (silica gel 1,2-dichloroethane-methanol,

6535 by volume). (N-hexyl-L-valyl)-cyclopeptide A hydrochloride N (forthe base) 12.25 (theory: 12.27) Rf= 0.37 (silica gel l,2-dichloroethanemethanol,

82 by volume). (N-benzyl-N-methyl-L-leucyl)-cyclopeptide A N 1 1.99(theory: 11.91) R1 0.72 (silica gel l,2-dichloroethane-methanol,

82 by volume).(44p-methoxybenzyloxycarbonylamino-cyclohexylcarbony1)-cyclopeptide A N10.8 (theory: 11.22) Rf 0.76 (silica gel 1,2-dichloroethane-methanol,

82 by volume). [(-methyl-3-phenthiazinyl)acetyl]-cyclopeptide A N 11.3(theory 11.56) S 2.53 (theory:

2.64) Rf 0.56 (silica gel 1,2-dichloroethane-methanol,

82 by volume). [2-( 3-benzoylphenyl)propionyl1-cyclopeptide A N 10.45(theory: 10.55) Rf 0.57 (silica gel methanol, 82 by volume).(N,Ndiheptylglycyl)-cyclopeptide A N 11.43 (theory: 11.56) Rf 0.74(silica gel 1,2-dichloroethane-methanol,

82 by volume).

EXAMPLE 3 I N-t.-butyloxycarbonyl-L-valine (0.21 g.) is dissolvedl,2-dichloromethanein methylene chloride (25 cc.) to which triethylamine(0.14 cc.) has been added. The mixture is cooled to 7 C., ethylchloroformate (0.1 cc.) is added and the mix ture then stirred for 35minutes at 5 C. A solution of cyclopeptide A (0.96 g.) in methylenechloride (10 cc.), to which triethylamine (0.14 cc.) has been added, isthen added. The mixture is stirred for 18 hours with external cooling bymeans of an ice bath, and concentrated to dryness under reduced pressure(25 mm.llg). Crude (N-t.-butyloxycarbonyl-L valyl)-cyclopeptide A (0.97g.) is obtained in an 84 percent yield. The product is dissolved inethyl acetate. The solution is filtered and then washed with water, andconcentrated to dryness under reduced pressure (25 mm.l lg). Pure (N-t.-butyloxy-carbonyl-L-valyl)-cyclopeptide A (0.73 g.) is thus obtainedin a 63 percent yield.

N 11.7 (theory: 12.1)

Rf= 0.70 (silica gel l,2-dichloroethane-methanol,

82 by volume).

EXAMPLE 4N-a-L(+)-6-methyloctanoy1-N-y-benzyloxycarbony1-L-a,'y-diaminobutyryl-L-threonyl-N-y-benzyloxycarbonyl-L-a,y-diaminobutyrylhydrazide(2.5 g.) [prepared according to the method described by K. Vogler etcoll, l-lelv. 48, 1161 (1965)] is dissolved in glacial acetic acid cc.)and N hydrochloric acid (7.08 cc.). The mixture is cooled to +2 C. andan icecold solution of sodium nitrite (0.232 g.) in water (2.5 cc.) isadded. The mixture is stirred for 15 minutes at 0 C., poured into aseparating funnel cooled to 0 C., and an ice-cold 5 percent solution ofsodium bicarbonate (200 cc.) is then added. The organic phase isdecanted and successively washed 5 times with an ice-cold 5 percentsodium bicarbonate solution (60 cc.). The washing liquors are againextracted with ice-cold ethyl acetate cc.). The organic extracts arethemselves washed with an ice-cold 5 percent solution of sodiumbicarbonate. All the organic phases are combined and then dried overanhydrous sodium sulphate at a temperature of between 0 C. and 2 C. Thesolution is filtered rapidly, and the solution of Na-L(+)-6-methyloctanoyl-N-y-benzyloxycarbonyl-L-a;ydiaminobutyryl-L-threonyl-N--y-benzyloxycarbonyl-L-a,'y-diaminobutyrylazide thus obtained is added to an ice-cold solutionof cyclopeptide A (3.23 g.) in ethyl acetate (100 cc.) to whichtriethylamine (0.47 cc.) has been added. The mixture is stirred for 18hours at a temperature of between 0 C. and 2 C., and then for 48 hoursat 20 C.

7 The r esulting solution is concentrated to dryness under reducedpressure (25 mm.l-lg). The residue is taken up in water (100 cc.) andtriturated until the residue becomes a powder. The mixture is filteredand then dried under reduced pressure (0.3 mm.l-lg) in the presence ofphosphorus pentoxide. The resulting product is taken up in acetone (20cc.). A slight amount of insoluble matter is filtered off and theliltrate concentrated to dryness under reduced pressure 20 mm.l-lg).

(N-a-L-(+)-6-methyl-octanoyl-N--y-benzyloxycarbonyl-L-a,-y-diaminobutyryl-L-threonyl-N-y-benzyloxycarbonyl-L-a;y-diaminobutyryl)-cyclopeptideA (4.44 g.) is thus obtained in a 79 percent yield.

N 11.54 (theory: 11.75)

Rf 0.92 (silica gel :1,2-dichloroethane-methanol,

l-l by volume).

The following products are prepared in the same manner, starting withappropriate starting materials: (N-a-palmitoyl-N-e-benzyloxycarbonyl-L-lysyl)- cyclopeptide A N 10.51(theory 10.56) C 64.65 Theory: 5

65 .03) H 8.89 (theory 9.05) Rf 0.75 (silica gel1,2-dichloroethane-methanol,

8-2 by volume).

(N-a-pelargonyl-N-'y-benzyloxycarbonyl-L-lysyl)- cyclopeptide A N 11.08(theory 11.32) C 63.18 (theory:

63.55) H%=8.41(theory:8.66) l EXAMPLE 5 Following the procedure ofExample 4 and starting withN-a-L(+)-6-methyloctanoyl-N--y-benzyloxycarbonyl-L-a,y-diaminobutyryl-L-threonyl-N-y-benzyloxycarbonyl-L-a,'y-diaminobutyrylhydrazide(2.5 g.), glacial acetic acid (75 cc.), N hydrochloric acid (7.08 cc.),sodium nitrite (232 mg. dissolved in 2.5 cc. of water),(N-methyl-L-valyl)-cyclopeptide A (3.6 g. dissolved in 100 cc. of ethylacetate to which 0.94 cc. of triethylamine has been added),(N-oz-L(+)-6-methyloctanoyl-N-'y-benzy1oxycarbonyl-L-a,y-diaminobutryrl-L-threonyl-N-y-benzyloxy-carbonyl-L-a,y-diaminobutyryl-N-methyl-L-valyl)-cyclopeptideA (5.23 g.) is obtained in an 87.5 percent yield. This product ispurified by chromatography over silica gel. There are used 50 g. ofsilica gel per 5 g. of product, and the product is eluted with ethylacetate and with a 9-1 (by volume) mixture of ethyl acetate-methanol.This purification results in chromatographically pure N-a-L(+)-6-methyloctanoyl-N-'y-benzyloxycarbonyl-L-aqdiaminobutyryl-L-threonyl-N-y-benzyloxycarbonyl-L-a,-y-diamino-butyryl-N-methyl-L-valyl)-cyclopeptide A (2.81 g.) in a 47percent yield.

N 11.79 (theory =11.79)

Rf 0.31 (silica gel 1,2-dichloroethane-methanol,

8-2 by volume). The following products are prepared in the same manner:(N-stearoyl-L-valyl)-cyclopeptide A N 10.46 (theory 10.58) I Rf= 0.45(silica gel 1,Z-dichloroethane-methanol,

8-2 by volume). (N-stearoyl-N-methyl-L-valyl)-cyc1opeptide A N% 10.02(theory: 10.4)

Rf= 0.78 (silica gel l,2-dichloroethane-methanol,

8-2 by volume).

7' 7' EXAM1 LE 6 l-leptanoic acid (1.43 g.) is dissolved indimethylformamide (5 cc.) and N-hydroxysuccinimide (0.173 g.) is thenadded. The mixture is cooled to 10 C. and dicyclohexylcarbodiimide (0.31g.) is added. The mixture is stirred for one hour at -10 C., then for 2hours at 0 C. and for 18 hours at 20 C.

The dicyclohexylurea formed is filtered off and a solution ofcyclopeptide A (1.44 g.) in dimetylformamide cc.), to whichtriethylamine (0.21 cc.) has been added, is then added to the filtrate.The mixture is then stirred for 18 hours at 20 C.

The reaction mixture is concentrated to dryness under reduced pressure(25 mm.Hg). The resulting residue is taken up in ethyl acetate (50 cc.)and successively washed with a 5 percent aqueous solution of sodiumbicarbonate, with N hydrochloric acid and with water. The organic phaseis dried over anhydrous sodium sulphate, filtered, and the filtrate thenconcentrated to dryness under reduced pressure (25 mm.l-lg).

The residue is chromatographed on twenty times its weight of silica gel,eluting with mixtures of chloroform-methanol of which the methanolcontent is progressively increased. The heptanoyl-cyclopeptide A iseluted with a 98-2 (by volume) mixture of chloroform-methanol. Thecorresponding fractions are concentrated to dryness under reducedpressure (25 .mmJ-lg) to give heptanoyl-cyclopeptide A (0.92 g.) in

a 37 percent yield.

N 11.2 (theory 11.77) C =62.6 (theory 62.83) H%=8.9 (theory:8.94) Rf0.82 (silica gel 1,Z-dichloroethane-methanol,

65-35 volume).

The following products are prepared in the same manner, starting withappropriate starting materials: stearoyl-cyclopeptide A C 65.80 (theory65.70) H 9.60 (theory 9.62) Rf 0.82 (silica gel1,2-dichloroethane-methanol,

65-35 by volume). n-butyryl-cyclopeptide A C% 60.9 (theory 61.9) H% 8.7(theory 8.72) Rf 0.55 (silica gel l,2-dichloroethane-methanol,

65-35 by volume). 4 sorboyl-cyclopeptide A N% 1 1.99 (theory: 11.97) Rf0.75 (silica gel 1,2-dichloroethane-methanol,

65-35 by volume). (1 1-1'-pyrrolidinyl-undecanoyl)-cyclopeptidehydrochloride N% 10.9 (theory 2.87) Rf 0.60 (silica gel 11,2-dichloroethane-methanol,

65-35 by volume). (2-1 '-pyrrolidinyl-hexanoyl)-cyclopeptidehydrochloride N% 11.81 (theory: 12.05 Rf 0.70 (silica gel1,Z-dichloroethane-methanol,

65-35 by volume).

EXAMPLE 7 Cyclopeptide A (2.875 g.) is dissolved in dimethylfonnamide(10.5 cc.) and 2,4,5-trichlorophenyl N-t.-

11.35) Cl 2.65 (theory butyloxycarbonyl-L-methionate (1.28 g.) is thenadded. The solution is cooled to C., triethylamine (0.42 cc.) is thenadded slowly and the mixture stirred for 18 hours at 0 C. The solutionis poured into a mixture of ice (45 g.) and cyclohexane (15 cc.) towhich acetic acid (0.02 cc.) has been added. A white oil is obtainedwhich is decanted and taken up in ethyl acetate (6 cc.). On addingpetroleum ether (100 cc.), a white solid is obtained which is filteredoff and dried to give (N-t.-butyloxycarbonyl-L-methionyl)-cyclopeptide A(2.85 g.).

This product (1.2 g.) is taken up in a 1.8N solution of hydrogenchloride in dioxan (2.73 cc.) and kept at 20 C. for hours. The mixtureis concentrated to dryness under reduced pressure (25 mm.i-lg). Theresulting product is taken up in ethyl acetate and then chromatographedover silica gel (15 g.) contained in a column 1.2 cm. in diameter. Thefractions eluted by a mixture of ethyl acetate-methanol (90-10 byvolume) are combined. The solvent is evaporated under reduced pressure(25 mm.Hg). The residue is taken up in water (50 cc.) and the resultingsolution is lyophilised to give (L- methionyl)-cyclopeptide Ahydrochloride (0.52 g.).

S%=2.92 (theory 2.84)

Rf 0.47 (silica gel 1,2-dichloroethane-methanol,

8-2 by volume).

The following product is obtained in the same way starting with theappropriate starting materials: (6-1'-pyrrolidinyl-hexanoyl)-cyclopeptide hydrochloride N 12.05 (theory:12.05)

Rf 0.52 (silica gel 1,2-dichloroethane-methanol,

65-35 by volume).

EXAMPLE 8 Cyclopeptide A (2 g.) is dissolved in methylene chloride (40cc.) and triethylamine (0.28 cc.) is then added. The mixture is cooledto a temperature of between 5 C. and -10 C. and the following twosolutions are then added simultaneously over the course of 10 minutes:

a. triethylamine (0.56 cc.) dissolved in methylene chloride cc.):

1 l-diethylamino-undecyl chloroformate hydrochloride (0.684 g.) inmethylene chloride (15 cc.).

The mixture is stirred for 2 hours at 0 C. and then for 18 hours at 20C. The reaction mixture is then washed with a 5 percent solution ofsodium bicarbonate followed by a saturated solution of sodium chloride.The mixture is dried over sodium sulphate, filtered and concentrated todryness under reduced pressure mm.Hg). The resulting residue ischromatographed over silica gel (25 g.) contained in a column 1.2 cm. indiameter. The fractions eluted with 98-2 and 95-5 (by volume) mixturesof ethyl acetate-methanol are combined and concentrated to dryness underreduced pressure (25 mm.Hg). The residue is taken up in water (40 cc.)and N hydrochloric acid is then added drop-wise until pH 3 is reached. Aslight amount of in soluble matter is filtered off and the filtrate islyophilised. 1 1-Diethylamino-undecyloxycarbonyl)- Cyclopeptide Ahydrochloride (1.24 g.) is thus obtained in a 50 percent yield.

N 10.87 (theory 11.07)

Rf 0.64 (silica gel 1,2-dichloroethane-methanol,

-35 by volume). The following product is prepared in the same manner:decyloxycarbonyl-cyclopeptide A C 63.50 (theory: 63.08) H 9.10 (theory:

9.08) N 11.95 (theory: 11.03) Rf 0.85 (silica gel1,2-dichloroethane-methanol,

8-2 by volume).

EXAMPLE 9 Cyclopeptide A (2.98 g.), o-nitrophenylsulphenyl chloride(0.569 g.) and triethylamine (0.42 cc.) are mixed in ethyl acetate (40cc.) at 2 C. The mixture is stirred for 18 hours with external coolingby means of an ice-bath and is then concentrated to dryness underreduced pressure (25 mm.Hg). A crude product (3.45 g.) is obtained andthis is chromatographed over silica gel (30 g.) contained in a column 2cm. in diameter. The fractions eluted with ethyl acetate areconcentrated to dryness under reduced pressure (25 mm.l-lg) to give(o-nitrophenylsulphenyl)-cyclopeptide A (1 g.).

N 12.1 (theory: 12.6) S ==2.85 (theory 2.88)

Rf 0.79 (silica gel 1,2-dichloroethane-methanol,

8-2 by volume).

The following products are prepared in the same manner, starting withappropriate starting materials:l-dimethylamino-S-naphthalenesulphonyl)cyclopeptide A N 11.66 (theory11.75) S 2.69 (theory:

2.69 Rf 0.70 (silica gel l,2-dichloroethane-methanol,

8-2 by volume). toluene-p-sulphonyl-cyclopeptide A l N 11.10 (theory:11.33) S 2.56 (theory:

2.88) Rf 0.74 (silica gel 8-2 by volume).p-acetylaminobenzenesulphonyl-cyclopeptide A N 11.71 (theory: 12.12) S2.93 (theory:

2.77) Rf 0.82 (silica gel 1,2-dichloroethane-methanol,

8-2 by volume).

EXAMPLE l0 (N-a-Acetyl-N-y-benzyloxycarbonyl-L-lysyl)- cyclopeptide A(863 mg.), prepared as described in Example 2, is dissolved in methanol(25 cc.). Palladium on charcoal (863 mg.; containing 3 percent of active1,2-dichloroethane-methanol,

metal) is added. The mixture is hydrogenated with violent agitation for2 hours at 20 C. and under a pressure of760 mm.Hg.

The resulting solution is filtered and the precipitate is washed with0.5N hydrochloric acid (10 cc.). The filtrate and the washing liquorsare combined and evaporated to dryness under reduced pressure (25 mm.Hg)in a rotating evaporator. The residue is dissolved in acetone (5 cc.)and the product is precipitated by adding ice-cold ether (50 cc.). Aftercooling for three hours at +4 C., the precipitate is filtered off,washed with ether and then dried for 18 hours under reduced pressure(0.3 mm.Hg) in the presence of phosphorous pentoxide to give(N-aacetyl-L-lysyl)-cyclopeptide A (0.46 g.) in a 57.6 percent yield.

N 13.0 (theory: 13.23)

[a9 =-77.2 (c 0.5, methanol) Rf 0.56 (silica gell,2-dichloroethane-methanol,

1-1 by volume).

The following products are prepared in the same manner, starting withappropriate starting materials: L-valyl-cyclopeptide A methanesulphonateN 11.83 (theory: 12.14)

Rf 0.55 (silica gel l,2-dichloroethane-methanol,

EXAMPLE 1 l(4-p-Methoxybenzyloxycarbonylamino-cyclohexylcarbonyl)-cyclopeptide A(0.5 g.), prepared by the method of Example 2, is dissolved in dioxancc.); a.

4N solution of anhydrous hydrogen chloride in dioxan (0.25 cc.) is addedand the resulting solution is stirred for 18 hours. The solvent isremoved under reduced pressure (30 mm.Hg) at 35 C. and the residue istaken up in ethyl acetate (20 cc.). The resulting solution is washedwith water (20 cc.) and the organic phase is decanted and evaporated todryness under reduced pressure (30 mm.Hg) at 35C.

The residue is taken up in dioxan (10 cc.) and treated with a 4Nsolution of anhydrous hydrogen chloride in dioxan (0.84 cc.) over aperiod of 4 hours. The solvent is removed under reduced pressure (30mm.Hg) at 35 C. and the residue is taken up in ethyl acetate 20 cc.).The solution is washed with water (20 cc.). The aqueous phase isdecanted and added to the aqueous phase obtained during the firsttreatment. The mixture is concentrated to dryness under reduced pres-65-35 by volume). (N-a-L(+)-6-methyloctanoyl-L-a,y-diaminobutyryl-L-threonyl-L-a,y-diaminobutyryl)-cyclopeptide A hydrochloride Cl 5.08(theory 4,81) Rf 0.14 (silica gel 1,2-dichloroethane-methanol,

l-l by volume).

( N-a-L(+ -6-methyloctanoyl-L-a,y-diaminobutyryl-L-threonyl-L-oqy-diamino-butyryLN-methyl-L-valyl)- cyclopeptide Ahydrochloride N 13.37 (theory 13.25) Cl 4.47 (theory Rf 0.05 (silica gell,Z-dichloroethane-methanol,

1-1 by volume). (N-a-palmitoyl-L-lysyl)-cyclopeptide A N 10.9 (theory:11.3) Rf 0.14 (silica gel l,2-dichloroethane-methanol,

8-2 by volume). (N-a-pelargonyl-L-lysyl)-cyclopeptide A hydrochloride N12.20 (theory: 12.19) Cl 2.95 theory:

Rf 0.08 (silica gel l,2-dichloroethane-methanol,

82 by volume). (N-a-L(+)-6-methyloctanoyl-L-a,y-diaminobutyryl)-cyclopeptide A hydrochloride N 12.27 (theory: 12.48) Rf 0.40 (silica gell,2-dichloroethane-methanol,

82 by volume). (N-methyl-p-dimethylamino-DL-pheny1alanyl)- cyclopeptideA N 13.05 (theory: 13.25) Rf 0.5 (silica gel1,2-dichloroethane-methanol,

82 by volume).

sure (0.3 mm.Hg) at 35 C. The residue is taken up in water 10 cc. aslight amount of insoluble matter is filtered off and the filtrate isevaporated to dryness under reduced pressure as above. The residue istaken up in water (10 cc.) and lyophilised. (4-Amino-cyclohexylcarbonyl)-cyclopeptide A (0.355 g.) is thus obtained in a 78 percent yield.

N 12.75 (theory: 12.51)

Rf= 0.43 (silica gel 1,2-dichloroethane methanol,

82 by volume).

EXAMPLE l2 Cyclopeptide A (10 g.) is dissolved in methylene chloride(200 cc.) and triethylamine (1.4 cc.) is added. The solution thusobtained is cooled to 5 C. and

thereafter an ice-cold solution of trichloroacetyl.

chloride (1.81 g.) in methylene chloride (20 cc.) is added over thecourse of 10 minutes. The reaction mixture is stirred for 2 hours at -5C. and then for 18 hours at ambient temperature. The solvent is removedby distillation under reduced pressure (30 mm.Hg) at 50 C. and theresidue is taken up in ethyl acetate cc.). Insoluble matter is filteredoff and the filtrate is successively washed with an ice-cold 5 percentsolution of sodium bicarbonate (2 X75 cc.), with N hydrochloric acid (2X 75 cc.) and then with a saturated solution of sodium chloride (30cc.). The organic phase is dried over sodium sulphate, filtered, and thefiltrate concentrated to dryness under reduced pressure (30 mm.Hg) at 50C. A crude product (7.23 g.) is thus obtained which is taken up in ethylacetate (20 cc.) and charged into a column 2 cm. in diameter containingsilica gel g.). Elution is eiTected with ethyl acetate. The product isrecovered in the fractions between and 450 cc. of eluate after removingthe solvent under reduced pressure (30 mm.Hg) at 50 C. After drying at30 C. under reduced pressure (0.1 mm.Hg), trichloroacetyl-cyclopeptide A(6 g.) is obtained in a 53 percent yield.

N%= 10.9 (theory: 11.13)

Rf 0.8 (silica gel 1,2-dichloroethane-methanol,

65-35 by volume).

EXAMPLE l3 N-Dodecyl-N-methyl-D-valine (65 g.) is dissolved in methylenechloride (800 cc.) and triethylamine (51 cc.) is added. The reactionmixture is cooled in an icebath, and cyclopeptide A (182 g.) is addedfollowed by dicyclohexylcarbodiimide (42.5 g.). After stirring for 18hours at 2- C. and then for 48 hours at a temperature of about 20 C.,glacial acetic acid (5 cc.) is added and the precipitate formed isfiltered off. The filtrate is concentrated to dryness. under reducedpressure (25 mm.Hg) and the residue is taken up in ethyl acetate (500cc.); the resulting suspension is kept in an icebath for 30 minutes andthe insoluble matter filtered off. The filtrate is concentrated todryness under reduced pressure (25 mm.Hg).

The crude product thus obtained is purified by chromatography. For thispurpose a solution of the crude product in ethyl acetate (500 cc.) ispoured through a column 10 cm. in diameter containing silica (2 kg.).Elution is effected with mixtures of ethyl acetatemethanol withincreasing proportions of methanol. Fractions of 300 cc. are collected.The fractions eluted philised to give (N-dodecyl-N-methyl-D-valyl)-cyclopeptide A hydrochloride (74 g.).

c 62.7 (theory: 63.06) H 9.25 (theory:9.25) CL 2.6 (theory:2.77) Rf=0.51 (silica gel:1,2-

dichloroethanemethanol, 8-2 by volume) EXAMPLE 14 The procedure ofExample 13 is followed but using N-undecyl-N-methyl-D-valine (1.75 g.)in methylene chloride (1 cc.), triethyl-amine (1.52 cc.), cyclopeptide A(5.4 g.) and dicyclohexylcarbodiimide (1.12 g.). After chromatographythrough a column containing silica (120 g. there is obtained(N-undecyl-N-methyl-D- valyl)-cyclopeptide A (1.83 g.

C 64.0 (theory:64.68)

H 9.09 (theory:9.54)

Rf 0.63 (silica gel: 1,2-

dichloroethanemethanol, 8-2 by volume) EXAMPLE The procedure of Example13 is followed but using N-tetradecyl-N-methyl-Dvaline (4.8 g.) inmethylene chloride (27 cc.), triethylamine (2.4 cc.), cyclopeptide A(10.3 g.) and dicyclohexylcarbodiimide (2.09 g.). After purification ofthe crude product by chromatog raphy through a column containing silica(100 g.) and transformation into the hydrochloride, there is obtained(N-tetradecyl-N-methyl-D-valyl)-cyclopeptide A hydrochloride (2 g.).

N 10.5 (theory:l0.73)

Cl '36 2.56 (theory: 2.71)

Rf= 0.68 (silica gel: 1,2-

dichloroethanemethanol, 8-2 by volume) EXAMPLE 16 The procedure ofExample 13 is followed but using N-tridecyl-N-methyl-D-valine (1.4 g.)in ethyl acetate (80 cc.), triethylamine (1.12 cc.), cyclopeptide A (4g.) and dicyclohexylcarbodiimide (0.9 g.). After purification of thecrude product by chromatography through a column containing silica (50g.) and transformation into the hydrochloride, there is obtained (N-tridecyl-N-methyl-D-valyl)-cyclopeptide A hydrochloride (0.48 g.).

N 10.32 (theory: l0.85) Cl 2.77 (theory:2.82) Rf 0.75 (silica gel: 1 ,2-

dichloroethanemethanol, 8-2 by volume).

n The present invention further includes within its scope pharmaceuticalcompositions which comprise at least one cyclopeptide of general formulaI, or a nontoxic acid addition or quaternary ammonium salt thereof, inassociation with a pharmaceutical carrier or coating. The inventionincludes especially such compositions made up for oral, parenteral orrectal administration, or as an ointment.

Solid compositions for oral administration include compressed tablets,pills, dispersible powders and granules. In such solid compositions oneor more of the active substances is or are admixed with at least oneinert diluent such as sucrose, lactose, or a starch. The compositionsmay also comprise, as is normal practice, additional substances otherthan inert diluents, e.g., lubricating agents such as magnesiumstearate.

Liquid compositions for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirscontaining inert diluents commonly used in the art, such as water andliquid paraffin. Besides inert diluents such compositions may alsocomprise adjuvants, such as wetting and suspending agents, andsweetening, flavoring and perfuming agents.

The compositions according to the invention for oral administration alsoinclude capsules of absorbable material such as gelatin containing oneor more of the active substances with or without the addition ofdiluents or excipients.

Preparations according to the invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of suitable non-aqueous media are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. These compositions may also containadjuvants such as wetting, emulsifying and dispersing agents. They maybe sterilized by, for example, filtration through a bacteria-retainingfilter, by incorporation in the compositions of sterilising agents, byirradiation, or by heating. They may also be manufactured in the form ofsterile solid compositions, which can be dissolved in sterile water ,orsome other sterile injectable medium immediately before use.

The compositions for rectal administration are suppositories whichcontain, in addition to the active substance, excipients such as cacaobutter or suppository wax.

The percentage of active ingredient in the composi tions of theinvention may be varied, it being necessary that it should constitute aproportion such that a suitable dosage shall be obtained. Obviouslyseveral unit dosage forms may be administered at about the same time.The dose employed depends upon the desired therapeutic effect, the routeof administration and the duration of the treatment. The dose can bebetween 2 and 500 mg. per kilogramme animal body weight. In humantherapy, the daily dose is generally between 0.1 and 10 g. of activeproduct.

The following Example illustrates pharmaceutical compositions accordingto the present invention.

EXAMPLE 17 Tablets are prepared in the usual way having the followingcomposition:

(N-benzyl-N-methyl-DL-valyl) -cyclopeptide A methanesulphonate 0.500 g.wheat starch 0.150 g. colloidal silica 0.040 g. magnesium stearate 0.010g In the treatment of tuberculosis the medicament is administered,preferably orally, at doses generally between 1 and g. per day.

' I claim:

4. A cyclopeptide according to claim 1 wherein R is 1. A cyclopeptide ofthe formula: 10

R L lranSA miHyi-iibiihl ii intimfiiflbfirfi iihifi glyizineL-trans-i-methyl-proline D-N-motllyl-leucine L leucine Lproline-L-N-methy1-valine wherein R is N-benzyl-N-methyl-L-valyl,N-benzyL N-dodecyl-N-methyl-D-valyl, and its non-toxic acid adsarcosyl,N-benzyl-N-methyl-L-methionyl, N-hexyl-N- 20 dition salts.

methyl-L-valyl, N-dodecyl--N-methyl-D-valyl, N-un- UNITED STATES PATENTOFFICE- fiERTiFICATE 6F CORRECTKON Patent NO. 3 719,65 Dated March 6,973

GEORGES JOLLES Inventor (:9)

It is certified that error appears in the above-identified patent andthat said Letters Patent; are hereby corrected as shown below:

In the heading at [30] the "Foreign Application Priority Data", for thenumber of the second French application.

"68,44421",, read 68l44421-; for the data for the third foreignapplication, read Nov. 12,1969 Belgium Signed and sealed this 13th dayof November 1973.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. k RENE D. TEGTMEYER Attesting Officer ActingCommissioner of Patents USCOMM-DC 60376-P69 i U.S. GOVERNMENT PRINIING OFFICE! I967 0-366-J3l.

ORN PO-IOSO (10-69)

1. A cyclopeptide of the formula:
 2. A cyclopeptide according to claim 1wherein R'' is N-benzyl-N-methyl-L-valyl, and its non-toxic acidaddition salts.
 3. A cyclopeptide according to claim 1 wherein R'' isN-benzyl-N-methyl-L-methionyl, and its non-toxic acid addition salts.